PolymerChemistry

PAPER

Cite this: Polym. Chem., 2020, 11,439

Received 25th July 2019,Accepted 25th September 2019

DOI: 10.1039/c9py01106d

rsc.li/polymers

Synthesis of modifiable photo-responsivepolypeptides bearing allyloxyazobenzeneside-chains†

Wei Xiong,‡ Chong Zhang,‡ Xiaolin Lyu, Hantao Zhou, Wenying Chang, Yu Bo,Erqiang Chen,* Zhihao Shen * and Hua Lu *

Synthetic polypeptides are important protein mimics due to their similar primary and secondary

structures. Previously, the properties of polypeptides can be regulated by either introducing

N-carboxyanhydride (NCA) stimuli-responsive moieties directly or modifiable groups for post-polymeriz-

ation functionalization. Here we combine the two approaches and report the synthesis of photo-respon-

sive and modifiable helical poly(L-glutamate) bearing an azobenzene (Azo) and an alkene (“ene”) group in the

side chain, namely P(AzoEne-Glu)s. The polypeptides, prepared by the controlled ring-opening polymerization

of a novel monomer AzoEne-GluNCA, undergo rapid and high yield UV-triggered trans–cis isomerization of

the Azo moiety. However, this change in the primary structure does not lead to interruption of the helical

secondary structure. The rigid helical main-chain and pendent Azo mesogen together endow P(AzoEne-Glu)s

with liquid crystalline properties. Moreover, the “ene” group of P(AzoEne-Glu)s can be modified with thiols via a

UV-mediated thiol–ene reaction, or quantitatively transformed to azide via a novel anti-Markovnikov hydroazi-

dation approach. The newly generated azido-containing polymer can be further converted by click-type cyclo-

addition reactions. This work may streamline the rapid synthesis of diverse functional and stimuli-responsive

polypeptides that are potentially useful for self-assembly and liquid crystalline materials.

Introduction

Synthetic polypeptides are biodegradable and biocompatibleprotein mimics bearing a peptide main chain and various sec-ondary structures including α-helices and β-sheets.1,2 Owing torecent advances in controlled ring-opening polymerization(ROP) of α-amino acid N-carboxyanhydrides (NCAs), polypep-tides are playing increasingly important roles in biomedicalapplications such as drug delivery, gene delivery, tissue engin-eering, and long-acting protein therapeutics.3–9 To manipulatethe properties of synthetic polypeptides, many strategies havebeen rigorously and successfully applied in the past twodecades. One of the approaches is to introduce responsive build-ing blocks to the side chain of NCA, which gave rise to respon-sive polypeptides after ROP. Along this direction, many interest-ing polypeptides showing responsiveness to external stimuli(e.g., temperature, redox, pH, and light) have been developed.10

Taking photo-responsive polypeptides as examples, commonphoto labile and/or switchable groups such as azobenzene(Azo),11–13 coumarin,14 2-nitrobenzyl,15 spiropyran,16 and cinna-myl17 have been implemented in NCA monomers for polymeriz-ation. Thanks to the precise spatiotemporal and remote controlof the light stimulus, these materials hold great promise forapplications such as photo regulated gene/siRNA transfection,18

self-assembly,16,19–21 and photo patterning,22 etc. Another strat-egy capitalizes on post-polymerization modification forexpanded repertoires of functional polypeptides.23,24 Notableexamples include highly efficient “click” type reactions such ascopper(I)-catalyzed azide–alkyne cycloaddition (CuAAC),25

thiol–yne/ene reaction,26–29 Michael addition,30 methioninealkylation,31,32 redox transformation,33 and olefin metathesis.33

Despite the tremendous progress, however, there are still press-ing needs for novel functionalization methods and new struc-tures. Moreover, polypeptides simultaneously harnessing thepower of both stimuli-responsiveness and post-polymerizationfunctionalization are still sparse. To realize this goal, one mustintroduce both responsive and modifiable moieties to the NCAmonomers, which may raise considerable synthetic challengesin molecular design and monomer purification.

Among many stimuli-responsive building blocks, Azo is awidely used photo-sensitive moiety that undergoes a reversibletrans–cis transition under UV irradiation.34–37 Apart from its

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c9py01106d‡These authors contributed equally to this work.

Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science

and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of

Education, College of Chemistry and Molecular Engineering, Peking University,

Beijing 100871, China. E-mail: chemhualu@pku.edu.cn, zshen@pku.edu.cn,

eqchen@pku.edu.cn

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photo reactivity, Azo is also a famous liquid crystalline (LC)mesogen.38–46 We have previously introduced Azo and oligo(ethylene glycol) (EGx, x = 2, 4, and 6) to 4-aminophenylalaninederived NCAs, whose ROP has led to photo- and thermal-dualresponsive polypeptides P(EGx-Azo)s.

11 Because the Azo buildingblock is only one –CH2– group away from the backbone in thesepolymers, the trans form polymer exhibited relatively low helicity,which was later completely disrupted by the trans–cis transitionof Azo. Moreover, the polymers tend to form nanosized aggre-gates in various solvents due to their intrinsic amphiphilicity andrelative rigid core (backbone + Azo). We envision that oneapproach to fine-tune the physicochemical properties is byadjusting the relative distance between the rigid Azo componentand the polypeptide backbone.

Here, we report the synthesis of a novel L-glutamate-derivedNCA monomer bearing an Azo mesogen and a modifiablealkene group in the side chain, namely AzoEne-GluNCA(Scheme 1). The ROP of AzoEne-GluNCA affords polypeptidesP(AzoEne-Glu)s with a controlled molecular weight (Mn) andnarrow dispersity (Đ). P(AzoEne-Glu) demonstrates a reversiblyphoto-responsive switch in the primary structure but not thehelical secondary structure. Lastly, we show that P(AzoEne-Glu)can be derivatized via the well-established thiol–ene reactionor converted to an azido group utilizing a novel protocol thathas not been applied in polymer science previously.

Materials and methodsMaterials

4,4′-Hydroxyphenylazobenzylalcohol (Azo-OH) was synthesizedfollowing procedures described previously. Anhydrous N,N-di-methylformamide (DMF) and hexamethyldisilazane (HMDS)were purchased from Sigma-Aldrich (St Louis, MO, USA). All

other chemicals were purchased from commercial sources andused as received unless otherwise specified.

Synthesis of 4-((4′-allyloxy) phenylazo) benzyl alcohol (Azoene-OH)

A mixture of Azo-OH (10.0 g, 43.8 mmol), allyl bromide (6.9 g,56.9 mmol), K2CO3 (7.9 g, 56.9 mmol), and KI (99.6 mg,0.6 mmol) in anhydrous acetone (200 mL) was stirred undernitrogen for 24 h at 60 °C. The reaction mixture was filteredand the solution was evaporated to remove acetone undervacuum. The residue was dissolved in CH2Cl2 (500 mL),washed with brine (100 mL), dried over Na2SO4, and concen-trated under reduced pressure. The crude product was furtherpurified by recrystallization from ethanol to give a red yellowcrystal (Azoene-OH, 9.4 g, yield: 80%). 1H NMR (400 MHz,CDCl3) δ 8.00–7.80 (m, 4H), 7.49 (d, J = 8.4 Hz, 2H), 7.10–6.96(m, 2H), 6.08 (ddt, J = 17.2, 10.5, 5.3 Hz, 1H), 5.52–5.26 (m,2H), 4.77 (s, 2H), 4.62 (ddd, J = 12.8, 7.1, 5.6 Hz, 2H). 13C NMR(101 MHz, CDCl3) δ 161.09, 152.23, 147.07, 143.19, 132.76,127.46, 124.74, 122.80, 118.12, 114.99, 69.06, 64.92.

Synthesis of 4-((4′-allyloxy) phenylazo) benzyl chloride (Azo-Cl)

To an ice cooled solution of AzoEne-OH (8.0 g, 29.8 mmol) inanhydrous CH2Cl2 (250 mL) was added thionyl chloride(2.8 mL, 38.8 mmol) drop-wise over a period of 20 min andthe mixture was stirred under nitrogen at room temperatureuntil AzoEne-OH was almost consumed as monitored by TLC(∼6 h). After neutralization with saturated aqueous NaHCO3,the separated organic layer was dried over Na2SO4 and evap-orated off under reduced pressure. The residue was purifiedby column chromatography (petroleum ether/ethyl acetate,10 : 1, v/v) to afford AzoEne-Cl (6.3 g, 74%) as an orangesolid.

1H NMR (400 MHz, CDCl3) δ 7.89 (dd, J = 19.4, 8.7 Hz, 4H),7.51 (d, J = 8.3 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 6.08 (ddd,

Scheme 1 Synthesis of AzoEne-GluNCA and P(AzoEne-Glu).

Paper Polymer Chemistry

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J = 22.5, 10.5, 5.3 Hz, 1H), 5.52–5.26 (m, 2H), 4.69–4.58 (m, 4H).13C NMR (101 MHz, CDCl3) δ 161.27, 152.58, 147.05, 139.48,132.74, 129.36, 124.88, 122.94, 118.13, 115.02, 69.07, 45.81.

Synthesis of γ-((4′-allyloxy) phenylazo)-L-glutamate (AzoEne-Glu)

L-Glutamic acid copper(II) complex copper(II) salt tetrahydrate(GCS) was synthesized following procedures described pre-viously.47 To a stirred solution of GCS (3.3 g, 6.7 mmol) andL-glutamic acid (2.0 g, 13.4 mmol) in mixed N,N-dimethyl-formamide (DMF)/water (12/1.9 mL), was added 1,1,3,3-tetra-methylguanidine (3.4 mL, 2.7 mmol) slowly over a period of10 min. After all solids were dissolved (∼3 h), a solution ofAzo-Cl in DMF (9.6 mL) was added to the deep blue solution.The mixture was further stirred at room temperature for 48 h.The slurry was added with acetone (300 mL) and kept stirringvigorously overnight until a fine precipitate was collected bycentrifugation. Next, the precipitate was sonicated for 10 minin acetone and centrifuged to remove the solvent. Afterrepeating this sonication–centrifugation procedure threetimes, the black green AzoEne-Glu copper(II) complex was col-lected and dried under vacuum. The AzoEne-Glu copper(II)complex was then added into a stirred solution of EDTA diso-dium salts. The black green solid gradually turned yellow andthe solution turned blue. After 24 h of stirring, the solid wascollected by filtration and washed with large amounts of DIwater until the solid became yellow. The crude product wasfurther recrystallized with water/isopropyl alcohol (1 : 2 v/v),and lyophilized to give AzoEne-Glu (3.6 g, 46% yield).1H NMR(400 MHz, DMSO) δ 7.90 (d, J = 7.6 Hz, 2H), 7.86 (d, J =8.3 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 9.0 Hz, 2H),6.09 (ddd, J = 22.4, 10.5, 5.2 Hz, 1H), 5.38 (ddd, J = 13.9, 11.8,1.4 Hz, 2H), 4.70 (d, J = 5.2 Hz, 2H), 3.97 (t, J = 6.6 Hz, 1H),2.78–2.56 (m, 2H), 2.23–2.04 (m, 2H). 13C NMR (101 MHz,DMSO) δ 172.09, 170.64, 161.44, 152.04, 146.58, 139.17,133.57, 129.26, 125.07, 122.77, 118.40, 115.76, 69.03, 65.68,51.35, 29.71, 25.39.

Synthesis of γ-((4′-allyloxy) phenylazo)-L-glutamateN-carboxyanhydride (AzoEne-GluNCA)

AzoEne-Glu (1.0 g, 2.5 mmol) and triphosgene (276.3 mg,0.9 mmol) were suspended in dry THF (60 mL) under nitrogen.The suspension was stirred at 50 °C for 4 h to obtain a clearsolution. After the removal of the solvent under vacuum, thecrude product was purified by recrystallization from THF/hexane (10/60 mL) in a glove box to yield pure AzoEne-GluNCAas a yellow powder (0.8 g, 77% yield). 1H NMR (400 MHz,CDCl3) δ 7.91 (d, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.2 Hz,2H), 7.03 (d, J = 8.9 Hz, 2H), 6.30 (s, 1H), 6.08 (ddd, J = 22.5, 10.5,5.3 Hz, 1H), 5.39 (dd, 2H), 5.19 (s, 2H), 4.63 (d, J = 5.3 Hz, 2H),4.39 (t, J = 6.0 Hz, 1H), 2.62 (t, 2H), 2.30 (ddd, J = 17.9, 11.7,6.1 Hz, 1H), 2.14 (td, J = 14.1, 6.9 Hz, 1H). 13C NMR (101 MHz,CDCl3) δ 172.45, 169.27, 161.29, 152.75, 151.35, 147.02, 137.17,132.70, 129.04, 124.87, 122.87, 118.15, 115.01, 69.07, 66.69,57.09, 30.05, 26.98. ESI-MS: calculated m/z 423.4; found m/z 422.3[M − H]−.

General procedure for the polymerization of AzoEne-GluNCA

In a glovebox, AzoEne-GluNCA (100 mg, 0.2 mmol, 50 equiv.)was dissolved in DMF (2 mL), followed by the addition of theinitiator HMDS (9.6 μL × 0.5 M, 1.0 equiv.). The polymerizationsolution was stirred for 30 h at room temperature. Upon com-pletion of polymerization, acetic anhydride (9.1 μL, 20 equiv.)was added and the solution was stirred for another 2 h beforethe polymer was precipitated in diethyl ether. The obtainedpolymer was then sonicated for 5 min in diethyl ether and centri-fuged to remove the solvent. After repeating this sonication–cen-trifugation procedure three times, the yellow product P(AzoEne-Glu) was collected and dried under vacuum (yield ∼90%).

General procedure for the post-polymerization modificationvia thiol–ene reaction

P(AzoEne-Glu) (30.0 mg, 0.079 mmol “ene”), thiol molecules(7.9 mmol “thiol”), and DMPA (1.8 mg, 0.007 mmol) were dis-solved in DMF (5 mL). The vial was purged with nitrogen for10 min and sealed with a cap. The mixture was irradiated witha 365 nm UV lamp (1.0 W cm−2) overnight; to ensure completeconversion, the reaction was irradiated for another 6 h withthe addition of 2 mL deionized (DI) water and 1.8 mg DMPA.The product was dialyzed against DI water and lyophilized toafford a yellow solid (typical ene conversion 70–85%).

General procedure for the post-polymerization modificationvia hydroazidation reaction (Synthesis of P(AzoN3-Glu))

P(AzoEne-Glu) (15.0 mg, 0.04 mmol “ene”) and benziodoxole(27.0 mg, 0.04 mmol) were added to a 5 mL vial equipped witha stir bar. The vial was evacuated, backfilled with nitrogen,and sealed with a rubber septum. Next, CH2Cl2 (0.2 mL) wasadded via a syringe to dissolve all substrates in the vial. Next,H2O (18.0 μL, 0.04 mmol) and TMSN3 (237.0 µL, 0.4 mmol)were added to the reaction. The mixture was stirred overnightbefore it was dried under vacuum and redissolved in DMF. Theobtained solution was dialyzed against DI water and lyophi-lized to afford a yellow powder product (yield ∼95%).

General procedure for post-polymerization modification viaCuAAC reaction

In a glovebox, P(AzoN3-Glu) (16.9 mg, 0.04 mmol “azido”),1-hexyne (6.6 mg, 0.08 mmol), and PMDETA (28.0 μL, 0.20 mmol)were dissolved in DMF before CuBr (11.2 mg, 0.08 mmol) wasadded to the mixture. The reaction mixture was stirred at roomtemperature for 24 h before being quenched with 1 M HCl. Theproduct was purified by dialysis against HAc–NaOAc buffer (pH 5)to remove copper ions in the dialysis bag with a cutoff molarmass of 1000 g mol−1 for 3 d. The resultant solution was lyophi-lized to afford a yellow powder (yield ∼85%).

Results and discussion

The amino acid AzoEne-Glu containing an allyoxyazobenzenemoiety was derivatized from L-glutamate in three steps asshown Scheme 1. Briefly, the compound Azo-OH containing a

Polymer Chemistry Paper

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phenolic hydroxyl group was reacted with allyl bromide in thepresence of K2CO3 and KI to generate AzoEne-OH in ∼80%yield. Next, the benzyl hydroxyl of AzoEne-OH was chlorinatedusing SOCl2 to afford the compound AzoEne-Cl with high con-version. AzoEne-Cl was nucleophilically attacked by theγ-carboxylate of GCS to generate AzoEne-Glu in ∼46% yield. Allprecursors were comprehensively characterized (Fig. S1–S6†).The obtained AzoEne-Glu was then cyclized using triphosgeneto obtain the monomer AzoEne-GluNCA in anhydrous THF.The identity of AzoEne-GluNCA was verified by 1H and 13CNMR, mass spectrometry, and FT-IR as shown in Fig. 1 andFig. S7–S8.†

Next, we conducted hexamethyldisilazane (HMDS)-mediated ROP of AzoEne-GluNCA to produce homo-polypep-tides P(AzoEne-Glu). The conversion of the monomer in DMFwas monitored by FT-IR spectroscopy based on the decrease inthe intensity of the anhydride νCvO bands at 1858 and1783 cm−1. The polymerization was quenched by acetic anhy-dride and the purified polypeptides were analyzed with sizeexclusion chromatography (SEC). As shown in Table 1 (entries1–4), at a feeding monomer-to-initiator ratio (M/I) of 30/1,50/1, 75/1 and 100/1, the obtained molecular weight (Mn) ofP(AzoEne-Glu) was 9.9, 20.3, 29.3 and 37.7 kg mol−1, respect-ively. The obtained Mns all agreed well with the expectedvalues. Moreover, all of the polymers showed a moderate dis-persity (Đ) of ∼1.16–1.26 (Table 1 and Fig. S9†).

P(AzoEne-Glu)s showed excellent solubility in commonorganic solvents such as DMF, THF, chloroform, and dichloro-methane. To study the photo-responsive behavior of P(AzoEne-Glu)100, we dissolved the polymer in CDCl3 and exposed thesolution to 365 nm UV irradiation for 15 minutes. Accordingto 1H NMR spectroscopy, the protons of Azo (Hb and Hc)showed a pronounced shift from 7.68–7.74 ppm to6.72–6.79 ppm after UV irradiation (Fig. 2B), indicating thetrans–cis transition of Azo with a calculated conversion of∼92%. Furthermore, exposure of the UV-treated P(AzoEne-Glu)100 to a blue LED lamp recovered the initial trans-form ofAzo with a conversion of ∼87%. The reversibility of P(AzoEne-Glu)100 was further confirmed by UV-Vis spectroscopy. TheP(AzoEne-Glu)100 solution in THF displayed a maximal absorp-tion peak at ∼350 nm associated with the π–π* transition oftrans-azobenzene. The intensity of this peak graduallydecreased after UV irradiation, along with a new adsorptionpeak emerging at ∼420 nm originating from the n–π* tran-sition of cis-azobenzene (Fig. 2C). Interestingly, we observed aslight increase in the elution time of P(AzoEne-Glu)100 from29.6 to 30.0 min after UV irradiation (Fig. 3D). This minorshift suggested a decreased hydrodynamic radius derived fromthe trans–cis transformation of Azo after UV irradiation.

Next, we investigated the secondary structure of P(AzoEne-Glu)100 in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solution bycircular dichroism (CD) spectroscopy. As shown in Fig. 3A, thetwo characteristic negative peaks at 208 and 222 nm in the CDspectrum illustrated a typical α-helical conformation of thepolypeptide. The helicity of the polymer was calculated to be∼71% based on the intensity at 222 nm. A small coupletadsorption band was observed at ∼325 and ∼375 nm, likelyderived from the cotton effect of π–π* transition of Azo,suggesting a small portion of ordered alignment of the pendantazobenzene (Fig. 3A, inset). Upon UV treatment, the 325 and375 nm peaks disappeared, which implied an order-to-disordertransition of the Azo moiety due to the trans-to-cis transition. Toour surprise, the characteristic polypeptide helix peaks at both208 and 222 nm did not show any sign of change, indicatingthat the α-helix structure remained stable even though the sidechain Azo switched from trans to cis conformation. This helicalstability was dramatically different from our previous work onP(EGx-Azo)s, which showed reversible switching in both theFig. 1 1H (A) and 13C NMR (B) spectra of AzoEne-GluNCA in CDCl3.

Table 1 Size exclusion chromatography (SEC) characterization ofP(AzoEne-Glu)n a

Entry Polymers Mn·cal.b Mn·obt

c Đd

1 P(AzoEne-Glu)30 11 400 9900 1.162 P(AzoEne-Glu)50 19 000 20 300 1.163 P(AzoEne-Glu)75 28 400 29 300 1.234 P(AzoEne-Glu)100 37 900 37 700 1.26

a Polymerizations were conducted in DMF at room temperature andcharacterized by SEC in DMF with 0.1 M LiBr. b Mn.cal. = calculatednumber-average molecular weight based on the feeding M/I ratio.c Mn.obt = obtained number-average molecular weight determined bySEC. dĐ = dispersity.

Paper Polymer Chemistry

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primary and secondary structures.11 We reasoned that the rela-tively long linker (–CH2CH2CO2CH2–) between the Azo andmain-chain in P(AzoEne-Glu), as compared to –CH2– in P(EGx-Azo)s, likely caused this consequence. We also tested the sec-ondary structure of P(AzoEne-Glu)100 in the solid state usingFT-IR spectra. The characteristic amide I and II absorbancebands at 1546 and 1653 cm−1 confirmed the α-helix ofP(AzoEne-Glu)100. No changes were observed after UV treat-ment, illustrating again the excellent conformational stability ofP(AzoEne-Glu)100 in the solid state (Fig. 3B).

To study the phase structure of P(AzoEne-Glu)50, we charac-terized the thermal properties of P(AzoEne-Glu)50 by thermo-

gravimetric analysis (TGA) and differential scanning calorime-try (DSC) in the first step. As shown in Fig. S10,† P(AzoEne-Glu)50 showed good thermal stability with the onset of weightloss (1%) temperature at ∼244 °C. In Fig. 4A, the DSC thermo-grams of P(AzoEne-Glu)50 showed an endothermic peak at∼140 °C during the heating process and an exothermic peak atalmost the same temperature during the reversed coolingcycle, which together suggested a phase transition. Next, thesolid state structure of P(AzoEne-Glu)50 was studied by wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering(SAXS). In the low angle region of SAXS, shown in Fig. 4B–Cand Table S1,† we observed four pairs of arcs on the equator at

Fig. 2 Photo-responsiveness of P(AzoEne-Glu)100. (A) Scheme of the UV-triggered trans–cis isomerization of P(AzoEne-Glu)100. (B) Overlay of 1HNMR spectra of P(AzoEne-Glu)100 in CDCl3 treated with UV (365 nm) and visible light irradiation; only the aromatic regions are shown for claritypurpose. (C) UV-vis spectra of P(AzoEne-Glu)100 in THF (c = 0.05 mg mL−1) treated with UV and visible light. (D) Overlay of SEC curves of P(AzoEne-Glu)100 before and after UV irradiation.

Fig. 3 Secondary structure of P(AzoEne-Glu)100. (A) CD spectra of P(AzoEne-Glu)100 in HFIP. (B) FT-IR spectra of P(L-AzoEne-Glu)100 in the solidstate. The polymers were first irradiated with UV at 365 nm for 5 min, followed by irradiation with a blue LED for 60 min.

Polymer Chemistry Paper

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q1 = 2.58 nm−1 (d spacing of 2.43 nm), q2 = 5.12 nm−1

(d spacing of 1.22 nm), q3 = 5.87 nm−1 (d spacing of 1.06 nm),and q4 = 7.67 nm−1 (d spacing of 0.82 nm). These four peakscorresponded to (200), (400), (410), and (600) reflections of a2D Colr structure with a = 1.78 nm, b = 4.86 nm, and γ = 90°.The schematic illustration of this phase structure at roomtemperature is shown in Fig. 4D. Because the combined lengthof the main chain and side chain under the all-trans confor-mation was 5.13 nm, most of the flexible linkers in the sidechains were expected to adopt gauche conformations.

Fig. 5 shows the 2D WAXD result of the sheared P(AzoEne-Glu)50 at room temperature with the shear direction on themeridian and X-ray beam along the z-direction. In the high-angle region, the four arcs (∼0.44 nm) marked by red ellipses

with the tilting angle at ±65° (α = 65°) in the quadrants(Fig. 5), which were easily distinguishable by angularintegration, revealed that the angle between the directions ofthe Azo and main chain was 65° (Fig. 4D). These four high-angle arcs were a consequence of the interference of the layerstructure composed of nematic-like Azo in each layer.Meanwhile, the two arcs (∼0.52 nm) marked by black sickleson the meridian, which were not observed in the 1D WAXDresult (Fig. S11†) but became clear in the 2D WAXD after shear-ing, corresponded to the interference between the helices ofthe main chain. The d spacing value was consistent with thehelical pitch of the helix (0.54 nm).

When P(AzoEne-Glu)50 was heated to 150 °C, five scatteringpeaks at q1′ = 2.53 nm−1 (d spacing of 2.48 nm), q2′ =4.64 nm−1 (d spacing of 1.35 nm), q3′ = 5.12 nm−1 (d spacing of1.22 nm), q4′ = 5.87 nm−1 (d spacing of 1.07 nm), and q5″ =7.67 nm−1 (d spacing of 0.82 nm) appeared in the low-angleregion (Fig. 4B and C), which corresponded to (200), (310),(400), (410), and (600) reflections of a 2D Colr structure witha = 2.16 nm, b = 4.96 nm, and γ = 90°. In the WAXD result, thediffraction peak at 2θ = 20.8° became a halo after heating(Fig. S11†). This change indicated the disordered orientationand isotropic state of the Azo. The schematic illustration ofP(AzoEne-Glu)50 at 150 °C is shown in Fig. 4E. It was hypoth-esized that the backbone of P(AzoEne-Glu)50 maintained a 2DColr structure regardless of the temperature variation, but itsAzo side chain experienced a anisotropy-to-isotropy phase tran-sition at increased temperature. The substantial increase ofa and b values (Fig. 4D and E) at 150 °C could be a result ofthe isotropic Azo. Similar results were also observed in othermain-chain/side-chain liquid crystalline polymers.48,49

Fig. 4 (A) DSC curves of P(AzoEne-Glu)50. (B) SAXS patterns of P(AzoEne-Glu)50 self-assembling at different temperatures. (C) Synchrotron SAXSpatterns of P(AzoEne-Glu)50 self-assembling at different temperatures. (D and E) Cartoon illustration of the top-view and side-view of the orderedstructure at room temperature (D) and the top-view of the structure at 150 °C (E) for P(AzoEne-Glu)50.

Fig. 5 2D-WAXD pattern of the sheared P(AzoEne-Glu)50 at roomtemperature with the shear direction on the meridian and X-ray beamalong the z-direction.

Paper Polymer Chemistry

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To further tune the properties of P(AzoEne-Glu), weexplored the post-polymerization modification of the “ene”moiety. Thiol–ene of P(AzoEne-Glu)100 with thiol moleculessuch as 1-hexanethiol, 2-aminoethanethiol, and sodium 2-mer-captoethanesulfonate was tested, affording polymers P(AzoC6-Glu)100, P(AzoNH2-Glu)100, and P(AzoSO3Na-Glu)100, respect-ively (Fig. 6A). Taking the reaction with 1-hexanethiol as anexample, the Hj peak at 5.99 ppm for P(AzoEne-Glu)100 almostdisappeared after UV irradiation (Fig. 6B, curve 2), indicatingan ∼88% conversion of alkene. An ∼60% and >95% graftingefficiency was achieved for P(AzoNH2-Glu)100 and P(AzoSO3Na-Glu)100, respectively (Fig. S12 and 13†). P(AzoC6-Glu)100 has agood solubility in most organic solvents such as DMF, THF,chloroform, etc. P(AzoNH2-Glu)100, however, dissolved poorlyin solvents such as THF, DCM, ACN, and neutral H2O. P(AzoNH2-Glu)100 was slightly soluble in acidic solvents such as1.0 M HCl aqueous solution and trifluoroacetic acid (TFA), likelydue to the incomplete modification of “ene”. P(AzoSO3Na-Glu)100had a relatively improved water-solubility compared to P(AzoNH2-Glu)100. Similar to the unmodified neutral P(AzoEne-Glu)100, theanionic polypeptide P(AzoSO3Na-Glu)100 showed an ∼68% heli-city, which was again found to be stable even after UV-triggeredAzo isomerization (Fig. S14†). This unusual helical stability wascomparable to that of those ionic helical polypeptides previouslydeveloped by Cheng et al.,50 underscoring again the importanceof the charge-backbone distance and aromatic building blocks tothe helical stability of synthetic polypeptides.

The poor solubility of P(AzoNH2-Glu)100 suggested intrinsicamphiphilicity, which stimulated us to explore its self-assem-

bly behavior. As such, we prepared an amphiphilic block co-polymer PEG5K-P(AzoNH2-Glu)100 by PEG amine (Mn = 5000g mol−1, PEG5K-NH2) initiated ROP of AzoEne-GluNCA and fol-lowed by thiol–ene modification with 2-aminoethanethiol(Fig. S15 and 16†). With the help of the hydrophilic PEG block,PEG5K-P(AzoNH2-Glu)100 dispersed into water and assembledinto micelles with an average particle size of ∼120 nm, measuredby dynamic light scattering (DLS, Fig. 6C; inset). The micellarstructure was further confirmed by atomic force microscopy(AFM), and the particle sizes of micelles measured by AFM andDLS agreed well as shown in Fig. 6C. When a less polar solventsuch as THF was used, we observed that PEG5K-P(AzoNH2-Glu)100 formed 1D nanowires with an average length of ∼3 μm(Fig. 6D and S17†). Moreover, when the polarity of the solventwas further decreased, PEG5K-P(AzoNH2-Glu)100 rapidly gener-ated an opaque organogel in DCM with a critical gelation concen-tration (CGC) of ∼3% (Fig. 6E; inset). The AFM image illustratedthe formation of a densely entangled 3D fibrous network(Fig. 6E). Although the exact mechanism is currently unclear,these results revealed that the microstructural dimension of theself-assembled PEG5K-P(AzoNH2-Glu)100 could be regulated bythe polarity of solvents.

Compared to alkyne/alkene-bearing polypeptides, azide-containing polypeptides are still rare. To our knowledge, onlythree examples of azide-containing NCAs were reported todate.25,51,52 It would be useful to switch between functional-ities so that mutually orthogonal click-like reactions can beimplemented later. According to a recent study by Xu et al.,the anti-Markovnikov hydroazidation of alkenes with TMSN3

Fig. 6 (A) Scheme of the thiol–ene reaction of P(AzoEne-Glu)100 with various small molecule thiols. (B) Overlay of 1H NMR spectra of P(AzoEne-Glu)100 before (top) and after (bottom) the thiol–ene reaction in TFA-d. (C–E) AFM images of the diblock co-polymer PEG5K-P(AzoNH2-Glu)100self-assembled in H2O (C), THF (D), and DCM (E).

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was realized using benziodoxole. However, its efficiency in thecontext of polymer has not been demonstrated.53 Inspired bythis work, we reacted P(AzoEne-Glu)100 with TMSN3 with thecatalysis of benziodoxole and water (Fig. 7A). As shown inFig. 7B, the peaks of alkene (Hj) at ∼1.80 ppm disappearedcompletely after the reaction, illustrated the full conversion of“ene” (middle). The obtained azide-containing polypeptide(P(AzoN3-Glu)100) was also verified by the characteristic absorp-tion band at 2090 cm−1 (νNvNvN) in FT-IR (spectrum 2 inFig. 7C). Moreover, the obtained P(AzoN3-Glu)100 was furthermodifiable with an alkyne molecule via a CuAAC reaction.Specifically, we reacted P(AzoN3-Glu)100 with 1-hexyne in thepresence of CuBr. The methylene protons adjacent to the azidemoiety (Hk) showed a significant shift from ∼3.5 to ∼4.5 ppm,demonstrating an almost quantitative transformation (bottom,Fig. 7B). This high efficiency was again confirmed by thedisappearance of the characteristic azido band in FT-IR(spectrum 3, Fig. 7C).

Conclusions

In conclusion, we designed and synthesized a novel monomerAzoEne-GluNCA bearing an allyloxyazobenzene moiety,

affording a photo-responsive and modifiable polypeptideP(AzoEne-Glu). 1H NMR, UV-vis, and CD spectroscopy showedthat the primary structure of the polypeptide experienced atrans–cis conversion; however, the helical structure remainedunexpectedly stable after UV treatment. The pendent Azo actedas a LC mesogen in the polypeptide. The LC phase ofP(AzoEne-Glu)50 formed with the help of the interplay betweenthe rigid helical main chain and pendent Azo. The pendent“ene” was modifiable with various thiol molecules via a thiol–ene reaction, and the resultant (co)polypeptides showed inter-esting assembly behaviours in a solvent-dependent manner.Moreover, the alkene moiety in the side chain was transformedto azide for a subsequent CuAAC reaction. Together, themonomer reported here could be a useful precursor and plat-form introducing various new structures. This work maystreamline the rapid synthesis of diverse functional polypep-tides that are potentially useful for self-assembly and liquidcrystalline materials.

Conflicts of interest

There are no conflicts to declare.

Fig. 7 (A) Scheme of the hydroazidation and subsequent CuAAC reaction of P(AzoEne-Glu)100. (B)1H NMR spectra of P(AzoEne-Glu)100 in TFA-d

before (top) and after hydroazidation (middle) and CuAAC reaction (bottom). (C) FTIR spectra of P(AzoEne-Glu)100 before (spectrum 1) and afterhydroazidation (spectrum 2) and CuAAC reaction (spectrum 3).

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Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (21434008 and 21722401).

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